Influenza A viruses (IAV) are significant human pathogens causing yearly epidemics and occasional pandemics. Past pandemics have resulted in significant morbidity and mortality. The 1918 influenza pandemic was thought to have resulted in the death of at least 675,000 people in the U.S., and 40 million people worldwide. Pandemics in 1957 and 1968, while less severe, were also of major public health importance. A novel influenza A virus of swine origin became pandemic in 2009, causing the first pandemic in 41 years. In addition, annual epidemic influenza causes are also very significant resulting in approximately 36,000 deaths in the US annually. Role of sialic acid binding specificity of IAV Hemagglutinin (HA) in virulence and pathogenesis: IAV HA is responsible for binding sialic acid containing glycoproteins on cells to initiate infection. The specificity of HA binding to terminal sialic acid (SA) residues is dependent on the conformation of the bond to the penultimate carbohydrate. In general, avian-adapted IAVs have a binding preference for SA alpha2,3 Gal and human-adapted IAVs have a binding preference for SA alpha2,6 Gal. To understand how changing HA binding specificity affects IAV virulence in a mouse model, we studied the growth, virulence and pathogenicity of a number of recombinant influenza viruses expressing wild-type and mutant avian and human HA genes created using reverse genetics on the backbone of a contemporary low passage human H1N1 virus. These studies revealed that the 1918 IAV HA sequence likely contains virulence motifs outside of the receptor-binding domain. In work this year, we are characterizing the sialic acid distribution by lectin histochemistry on respiratory tissues of various animal models of influenza, and developing methods to examine influenza virus binding to fixed tissue sections. The virulence of the 1918 influenza virus is still incompletely understood. To further evaluate the virulence encoded by the 1918 HA, we constructed a series of chimeric influenza viruses that contained the HA segment of the 1918, 1957, 1968 or 2009 pandemic influenza viruses in the context of 7 identical genomic segments derived from a seasonal H1N1 influenza strain. We analyzed the role of the influenza viral hemagglutinin (HA) protein in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the binding activity of HA proteins to lung surface surfactant protein D (SP-D), and their ability to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Pandemic HA-expressing viruses were evaluated for disease severity and pathology, viral replication, inflammatory responses, and SP-D binding compared to viruses containing recent seasonal HA segments in BALB/c mice. We found that viruses expressing the HA gene of all pandemic viruses were associated with significant lower respiratory tract pathology, and acute inflammation and showed low binding activity for SP-D. In contrast, viruses expressing the HA of seasonal influenza strains resulted in mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D. Low SP-D binding activity is thus a shared feature of the HA proteins of the past four pandemic influenza viruses and may be an important feature of future pandemic influenza viruses ability to cause severe disease. The role of the ribonucleoprotein complex in host adaptation and pathogenesis of IAV: The proteins that make up the IAV ribonucleoprotein (RNP) complex - PB2, PB1, PA, and NP are likely to play a significant role in pathogenesis of IAV infection since a virus that can replicate to a high titer may be more pathogenic, and mutations in these genes are also very likely to be crucial for host switch events in IAV since replication must occur in the context of the host cellular milieu. Because IAVs are known to acquire host-adaptive genome mutations, and since the PB2 gene of the 2009 H1N1 virus is of recent avian derivation, there exists concern that the pathogenicity of the 2009 H1N1 influenza A pandemic virus could be potentiated by acquisition of the host-adaptive PB2-E627K or -D701N mutations, which have been shown to enhance the virulence of other influenza viruses. We evaluated influenza viral infections in a mouse model which showed that these mutations did not increase the virulence of viruses containing the 2009 H1N1 viral polymerase. This further supports the importance of historical contingency in the development and evolution of novel influenza viruses. Epidemic or seasonal influenza is also a significant cause of illness and death. This year we characterized a novel in-frame deletion occurring in the neuraminidase gene of a recent H3N2 virus isolated from a patient on oseltamivir antiviral therapy. The virus was not attenuated in its growth in vitro or in vivo, and was efficiently transmitted in a ferret model system.